VDAC/porin is present in sarcoplasmic reticulum from skeletal muscle
VDAC/porin is present in sarcoplasmic reticulum from skeletal muscle
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DOI：
10.1016/0014-5793(96)00442-5
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发表时间：
1996-05-20
期刊：
影响因子：
3.5
通讯作者：
Heilmeyer, LMG
中科院分区：
文献类型：
--
作者：
ShoshanBarmatz, V;Hadad, N;Heilmeyer, LMG
在本研究中，我们使用多种方法证明了肌浆网 (SR) 中存在具有电压依赖性阴离子通道 (VDAC) 特性的蛋白质，总结如下： (a) 不同 SR 制剂中的 35 和 30 kDa 蛋白质，通过 Ca2+/草酸盐负载和 55% 蔗糖沉淀从其他膜系统中纯化，与四种不同的蛋白质发生交叉反应。 VDAC 单克隆抗体，(b) 源自 SR 35 kDa 蛋白的三种肽的氨基酸序列与 VDAC1 同工型中存在的序列相同，(c) 与线粒体 VDAC 类似，SR 蛋白由 [C-14]DCCD 特异性标记，(d) 使用新方法，从 SR 和 (e)重构为平面脂质双层后，纯化的SR蛋白显示出电压依赖性通道活性，其性质与来自人B淋巴细胞的纯化线粒体VDAC或VDAC1/孔蛋白31HL相似，并且其通道活性被阴离子转运抑制剂DIDS完全抑制，并且被DCCD抑制约80%， 我们还证明了 ATP 易位到 SR 腔中以及该 ATP 对腔蛋白肌钙蛋白的磷酸化，ATP 易位和肌钙蛋白磷酸化均受 DIDS 抑制，但不受白术苷（ATP/ADP 交换器阻断剂）的抑制。这些结果表明 VDAC 的存在，被认为仅位于线粒体中，在 SR 中 骨骼肌的结构及其可能参与 ATP 运输，再加上最近对 VDAC 多室定位及其与酶和通道的动态关联的研究，我们的研究结果表明，VDAC 作为一种有助于各种细胞功能的蛋白质值得关注和考虑。
In this study we demonstrate the existence of a protein with properties of the voltage-dependent anion channel (VDAC) in the sarcoplasmic reticulum (SR) using multiple approaches as summarized in the following: (a) 35 and 30 kDa proteins in different SR preparations, purified from other membranal systems by Ca2+/oxalate loading and sedimentation through 55% sucrose, cross-react with four different VDAC monoclonal antibodies, (b) Amino acid sequences of three peptides derived from the SR 35 kDa protein are identical to the sequences present in VDAC1 isoform, (c) Similar to the mitochondrial VDAC, the SR protein is specifically labeled by [C-14]DCCD, (d) Using a new method, a 35 kDa protein has been purified from SR and mitochondria with a higher yield for the SR, (e) Upon reconstitution into a planar lipid bilayer, the purified SR protein shows voltage-dependent channel activity with properties similar to those of the purified mitochondrial VDAC or VDAC1/porin 31HL from human B lymphocytes, and its channel activity is completely inhibited by the anion transport inhibitor DIDS and about 80% by DCCD, We also demonstrate the translocation of ATP into the SR lumen and the phosphorylation of the luminal protein sarcalumenin by this ATP, Both ATP translocation and sarcalumenin phosphorylation are inhibited by DIDS, but not by atractyloside, a blocker of the ATP/ADP exchanger, These results indicate the existence of VDAC, thought to be located exclusively in mitochondria, in the SR of skeletal muscle, and its possible involvement in ATP transport, Together with recent studies on VDAC multicompartment location and its dynamic association with enzymes and channels, our findings suggest that VDAC deserves attention and consideration as a protein contributing to various cellular functions.